Adaptive signaling of adenylyl cyclase (AC) has been implicated in a variety of neuropsychiatric and neurologic disorders including schizophrenia, drug abuse, and Parkinson's disease. Heterologous sensitization of AC is thought to play a primary role in this process. Acute activation of G?i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in sensitization of AC signaling and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of receptors including D2 dopamine and ? opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. Therefore, the overall objective of this research proposal is to use a non-biased approach and advances in target discovery to identify the molecular pathways involved in heterologous sensitization of three neuronal AC isoforms (i.e., AC1, AC2, and AC6). Each of the ACs show robust expression in the brain; however, their regulatory mechanisms (including sensitization) are unique. Despite their regulatory differences, previous studies have implicated phosphorylation, G?? subunits, and signalosome assembly as potential overlapping components in the molecular mechanisms of heterologous sensitization of AC. To identify both unique and overlapping genes associated with sensitization of AC, we propose to develop and validate a series of three scalable cAMP sensitization assays for genome-wide siRNA library screening. Cell lines co-expressing individual AC isoforms and the D2L dopamine receptor will be used for this effort. However, an important first step will be the development of robust cell-based assays that can be used for high throughput screening (HTS) in 384 well format. Each HTS assay will also be validated for reverse transfection with siRNA with appropriate positive and negative controls. The genome-wide siRNA library screening will be completed in collaboration with the Center for Chemical Genomics (CCG) at the University of Michigan. The newly identified genes will be then validated and further characterized using several cellular assays to assess specificity for AC isoforms, cellular background, and receptor type. We anticipate that the proposed studies will identify new protein targets that could ultimately be used to prevent the heterologous sensitization of AC/cAMP signaling that occurs in vivo. The results from the proposed experiments are likely to increase our understanding of adaptive changes that occur in central nervous system disorders, and the findings may also lead to improved treatment strategies.